Abstract Disturbed glucose homeostasis, particularly in skeletal muscle, is a fundamental contributor to type 2 diabetes. Despite the known importance of lipid metabolism in regulating glucose metabolism, the role of mitochondria in this regulation has been contentious. The objective here is to define the role of acyl-CoA thioesterase-2 (Acot2) in skeletal muscle lipid and glucose metabolism. Acot2 resides within the mitochondrial matrix and hydrolyzes long-chain fatty-acyl CoA. Its biological role is unknown. We hypothesize that Acot2 provides a safety valve to limit the flow of long-chain fatty acyl-CoA into ?-oxidation, thereby curbing the abundance of long-chain fatty acyl-CoA and acetyl-CoA in the mitochondrial matrix. Acetyl-CoA accumulation within muscle mitochondria has been linked to disturbed systemic glucose homeostasis and to a lesser ability to switch between glucose and lipid oxidation (metabolic flexibility) in muscle. Thus, we also hypothesize that Acot2 in muscle promotes substrate switching and contributes to glucose homeostasis. We base these hypotheses on Preliminary Studies from our lab showing that Acot2 dampens ?-oxidation and lipid-induced reactive oxygen species production, enables better matching between ?-oxidation and oxidative phosphorylation, and improves metabolic flexibility at the level of mitochondria and glucose homeostasis systemically. We have also observed that Acot2 is required for the full activity of carnitine acetyltransferase (CrAT) which was recently shown to be required for normal substrate switching in muscle and systemic glucose homeostasis. The rationale here is that identifying the biological role of Acot2 will provide new insight into the mechanisms by which mitochondria regulate skeletal muscle glucose disposal, and thus systemic glucose homeostasis. The hypothesis will be tested in three specific aims to define how Acot2: 1) influences substrate metabolism and uncoupled respiration in skeletal muscle mitochondria; 2) impacts glucose disposal and substrate switching in skeletal muscle and whether Acot9 compensates for Acot2 absence; 3) determines redox state of the mitochondrial matrix, and if perturbed mitochondrial redox influences glucose homeostasis when Acot2 is absent. Studies will utilize a new Acot2 loss-of-function model and CrAT gain-of-function, and will integrate observations from bioenergetics, radioisotope, metabolomics analyses and imaging experiments, on different muscle preparations, with tissue and whole-body metabolism. These studies are innovative because they approach skeletal muscle metabolism from an understudied pathway of mitochondrial long-chain fatty-acyl hydrolysis. The significance of studying this pathway is indicated by our findings that this pathway can broadly influence systemic glucose homeostasis.